It is known that transient high intensity electric fields can cause reversible, and at higher intensities, irreversible pores to form in lipid by-layers such as those that form membranes surrounding eukaryotic and prokaryotic cells. This principle forms the basis for a technique called "electroporation" which is of great and increasing importance to the emerging bio-technology industry. Of central importance to this industry is the process of "transformation" of cells in culture, a process whereby a cell is induced to express a gene whose product causes the cell to be commercially useful in some way. Currently, this usually means that the cell will produce a protein (e.g., insulin), which it does not normally produce, in large quantities. The protein is then harvested and purified for commercial purposes.
The process of transformation, reduced to its most basic level, requires two steps: 1) a sample of DNA containing the necessary sequences for the production of the desired product must be created, and 2) the DNA must be introduced into the living cells that are to be transformed. The second step involves a delicate compromise-the cell membrane must be weakened sufficiently to allow the DNA to diffuse into the cell, while not being weakened so much that the essential components of the cell leak out, thereby killing the cell.
Many methods have been employed to weaken cell membranes for the purpose of cell transformation, including physical shocks such as freezing, thawing, exposure to high temperatures or osmotic shock, chemical treatments such as detergents and calcium salts, and enzymatic treatments with proteolytic enzymes or lysozymes. These techniques all suffer from a common disadvantage-the damage they do to the cell membrane lasts until the cell actively repairs it, which many cells may never be able to accomplish. The result of this is that treatments sufficient to allow large amounts of DNA into the cells kill virtually all of the cells, while treatments mild enough to allow a large percentage of the cells to survive allow too little DNA to enter the cells.
The technique of electroporation overcomes this problem, because it generates holes or "pores" in the cellular membrane that last only a few milliseconds. This is because the pores are self-healing. Because of this, an electroporation resulting in a given cell survival allows much more DNA to enter the cells than any of the other techniques, thus achieving much higher transformation frequencies. In addition, electroporation has been shown to work in organisms highly resistant to transformation with any other method.
Electroporation has been used successfully on several eukaryotic cell types, including both animal and plant cell types. However, attempts to apply the technique to smaller cells, such as bacteria, have yielded uselessly low transformation rates. Since most commercial applications of recombinant DNA technology require that a gene be expressed in bacteria suitable of mass production, the development of a technique allowing the electroporation of bacterial cells is of great importance to the industry.
It is an object of the present invention to provide apparatus for the highly efficient transformation of living cells by the introduction of DNA thereto through transient pores created in the cell walls by electroporation.
It is a further object of the invention to provide apparatus for electroporating living cells with cell transformation efficiencies in excess of 10.sup.9 bacterial transformants per microgram of DNA.
Another object of the invention is to provide apparatus for the high efficiency transformation, by electroporation, of living bacterial, plant and animal cells in a suspension including DNA.
Yet another object of the invention is to provide a method for the transformation of living cells by the introduction of DNA thereto through transient pores created in the cell walls by the highly efficient electroporation of such cells in suspension with DNA.
Other objects and advantages of the invention will be apparent from the following detailed description of apparatus and methodology for accomplishing the highly efficient transformation of living cells by electroporation in accordance with the invention taken together the accompanying drawing figures.